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Halogenated Thymidine Analogues Restore the Expression of Silenced Genes without Demethylation

¹ Laboratory of Virus Immunology, Institute for Virus Research, Kyoto University, Kyoto, Japan; ² Department of Hematology, Kumamoto University School of Medicine; and ³ Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan

Requests for reprints: Masao Matsuoka, Laboratory of Virus Immunology, Institute for Virus Research, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-4048; Fax: 81-75-751-4049; E-mail: mmatsuok{at}virus.kyoto-u.ac.jp.

Transcriptional silencing of tumor suppressor genes by aberrant DNA methylation is a characteristic frequently observed in cancer cells. Therefore, reversing this process is a therapeutic target against cancer. In this study, we established a screening system for silencing inhibitors with cell lines transfected by a retroviral vector containing a luciferase gene. More than 100 nucleosides were tested for antisilencing activity with a selected clone in which the silenced expression of luciferase could be recovered by 5-aza-2'-deoxycytidine. A group of halogenated thymidine analogues was found to reactivate transcription of not only the reporter retrovirus vector but also endogenous glutathione-S-transferase 1 gene, without influence to DNA hypermethylation. Gel mobility shift assay showed that 5-bromo-2'-deoxyuridine (BrdUrd) or 5-iodo-2'-deoxyuridine incorporation did not affect the binding of the methyl-CpG binding protein motif to methylated DNA. Finally, in the retroviral promoter, BrdUrd treatment increased the acetylated histone H3 level and decreased methylation of histone H3 Lys⁹ in accordance with recovered transcription. This study shows that halogenated thymidines have an antisilencing effect without changing DNA methylation status by interfering with step(s) between DNA methylation and histone acetylation.

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